Picornaviruses (Picornaviridae) are a diverse family of viruses which cause a number of common illnesses. Of the Picornaviridae family, viruses of the genus Enterovirus are significant for a number of diseases and affect millions of people worldwide each year. Nonspecific febrile illness is one of the most common presentations of an enterovirus infection. Other notable diseases caused by an enterovirus infection include poliomyelitis, pleurodynia, pericarditis, myocarditis, arrhythmias, myocardial infarction, and acute haemorrhagic conjunctivitis.
Viruses of the genus Enterovirus are often found in the respiratory secretions (e.g. saliva, sputum, or nasal mucus) and stool of an infected person. Historically, poliomyelitis, which is caused by the 3 polioviruses, was the most significant disease caused by an enterovirus. However, there are now close to a hundred non-polio enteroviruses that can cause diseases in humans; these include Coxsackie A viruses, Coxsackie B viruses, echoviruses, and many other enteroviruses. Excluding rhinoviruses, enteroviruses that cause human disease have recently been re-classified into 4 distinct species. Polioviruses and enterovirus 71 (EV71), like many other enteroviruses, are spread through the fecal-oral route. Infection can result in a wide variety of symptoms ranging from mild respiratory illness (common cold), hand, foot and mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like disease, and acute flaccid paralysis.
Enterovirus infections are the most common causes of aseptic meningitis in children. In the United States, enteroviruses are responsible for 30,000 to 50,000 cases of meningitis. Further, a 2007 study suggested that acute respiratory or gastrointestinal infections associated with enteroviruses may be a factor in chronic fatigue syndrome.
Human Enterovirus 71 and Coxsackievirus A16 (CA16) are enterovirus serotypes in species A that are notable as the major causative agents for hand, foot and mouth disease (HFMD). The virus is excreted in faeces and is also found in pharyngeal secretions and blisters. Transmission is associated with close contact among children and through environmental contamination. The disease is characterized by an acute onset of fever with a rash on the palms, soles, buttocks, and knees, and vesicles on buccal membranes that usually resolves in 7-10 days. Only a small proportion of children with HFMD develop severe disease.
Some of the children infected by EV71 develop encephalitis, which is a rare manifestation of an enterovirus infection. Encephalitis may result in permanent brain damage and can be fatal. Severe disease involving primarily the neurologic and cardiovascular systems manifesting as syndromes such as meningitis, encephalitis, brainstem encephalitis, acute flaccid paralysis, pulmonary edema and cardiac failure generally occur only with EV71 infection. In the Asia-Pacific Region the most devastating neurological syndrome is brainstem encephalitis, which has a mortality rate of 40-80 percent. Children with severe HFMD may take months to recover, and in some cases the neurologic damage may be permanent. Currently, there is no specific antiviral treatment for HFMD and no vaccines to prevent enterovirus infection other than polio.
EV71 is additionally sometimes associated with severe central nervous system diseases. It was first isolated and characterized from cases of neurological disease in California in 1969. To date, little is known about the molecular mechanisms of host response to EV71 infection, but increases in the level of mRNAs encoding chemokines, proteins involved in protein degradation, complement proteins, and pro-apoptotic proteins have been implicated.
Although the virus has been detected worldwide since then, the recent regional epidemics of HFMD in Asia has raised concern that more pathogenic forms of EV71 may be emerging in the region. The first recognition of a HFMD outbreak with a high number of fatalities was in Sarawak, Malaysia in 1997. The virus associated with the outbreak then was EV71. Taiwan reported 129,106 HFMD cases in a 1998 epidemic with 405 having severe disease, and 78 deaths. Singapore reported an epidemic of 9000 cases with 7 deaths during 2000-2001, and since then has experienced recurrent epidemics every two to three years. During the first 8 months of 2008, Singapore reported 19,530 cases and one death due to HFMD. Since then EV71 outbreaks have been reported regularly in Singapore, Thailand, Malaysia, Taiwan, Japan, Korea, Vietnam, China, Australia and the Philippines.
China reported 83,344 cases with 17 deaths in 2007, and in 2008 experienced a large outbreak in Fuyang City in Anhui Province spreading throughout many parts of China. These large outbreaks were widely covered by the press, which highlighted parental concerns about the health of their children and the social disruption from closing of schools and day care centers by public health departments in an attempt to break the chain of transmission. Since then China has reported large outbreaks annually.
Monoclonal antibodies generated against peptides from EV71 capsid proteins generally have no or low neutralizing activity. Zhang et al., (2012) generated monoclonal antibodies against amino acid residues 94 to 97 which were reactive by western blotting, immunofluorescence and ELISA but could not neutralize EV71 virus. Kiener et al., (2012) described a monoclonal antibody against a VP2 capsid peptide (aa 142-146) which specifically reacts with EV71 but does not neutralize the virus. A commercially available monoclonal antibody MAB979 (Merck Millipore) which is reactive against a VP2 peptide (aa 141 to 150) (Liu et al., 2011) neutralizes the prototype EV71 (BrCr) at a titre of <1:14. Peptide SP55 (aa 163-177) which is within VP1 capsid and peptide SP70 (aa 208-222) also within VP1 generate monoclonal antibodies which either do not neutralize EV71 or do so at low titres such as 1:4 or 1:16 at best (Li et al., 2009). Lim et al., (2012) described 2 monoclonal antibodies generated to a peptide that is located within the SP70 peptide (Li et al., 2009) which share a common epitope KQEKD. One of the monoclonal antibodies neutralizes EV71 while the other does not. The authors suggest that the neutralization is dependent on antibody isotype, in this case, it is the antibody that has an IgM isotype that neutralizes while the IgG antibody does not. IgM has 10 binding sites while IgG has only 2, suggesting that the neutralizing activity is dependent on a blocking action of the antibody.
As the antibodies described above lack potency, there is a need to provide more effective monoclonal antibodies specific for EV71, and a method for producing potent neutralizing antibodies against EV71 and other picornaviruses. In addition, EV71 antibodies are needed for process controls and potency determination in EV71 vaccine development and would also be useful to help define EV71 antibody potency standards.